The tree-like architecture of the mammary gland is generated by branching morphogenesis, a reiterative process of branch site initiation and tubule invasion from a pre-existing epithelial structure. Branching is controlled by the interplay between positive and negative regulators, defects in either of which can give rise to aberrancies ranging from hyperplasia to malignant growth. Our long term goal is to delineate how these positive and negative signals are integrated spatially within the tissue to determine which cells branch, and thereby define the branching pattern. We have developed a lithography-based three-dimensional organotypic culture model that recapitulates the architecture of mammary epithelial ducts, enables micrometer-resolution control of tissue geometry and microenvironment, and provides quantitative data in a physiologically relevant context. The engineered ducts execute a complete series of morphogenetic events that can be predicted computationally. Using this culture model, we have shown that the position of branching is determined in part by the concentration profile of transforming growth factor (TGF)-21, an autocrine inhibitory morphogen. Furthermore, we have found that cells located in positions that branch up- regulate the expression of mesenchymal markers during morphogenesis. Based on these preliminary and published data, we propose: 1- To investigate the features of the TGF21 concentration profile perceived and transduced by mammary epithelial ducts. 2- To determine the mesenchymal markers differentially expressed during morphogenesis, and whether these are necessary and/or sufficient to define position of branching. We will further test whether the pattern of mesenchymal gene expression is regulated by the TGF21 inhibitory profile. 3- To begin to dissect how branching is regulated by the physical properties of the microenvironment, by determining whether the extracellular matrix alters branching pattern, TGF21 inhibitory concentration profile, or neo-expression of mesenchymal markers. These studies will provide insight into the local cues and gene expression changes that govern position of branching. PROJECT RELEVANCE: Cells integrate information from stimulatory and inhibitory signals during branching morphogenesis to develop into the tree-like structure of the mammary gland; disruption or misregulation of these signals can lead to neoplastic growths and eventual development of frank tumors. Here we present studies aimed at understanding how mammary epithelial cells perceive inhibitory signals and translate them into patterned differences in gene expression during branching morphogenesis.